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Objectives Stomach adenocarcinoma (STAD) is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. It is important to understand its molecular basis better and identify noninvasive biomarkers for targeted therapy. Usually, tumors contain driver genes or passenger genes, which can be regulated by microRNAs (miRNAs). However, the mechanism underlying the interaction between miRNA and their target genes in STAD has not been fully demonstrated. To date, no miRNA-targeted gene mutation sites that promote STAD progression have been reported, and there are no reports of the mutations within the 3′-UTR of COL12A1 that could contribute to STAD development. Methods Here, multiple genomic and transcriptomic profiles of STAD patients were downloaded from several reputable online databases and various analyses were conducted, including assessments of mutation rates, copy number variations (CNVs), single‐nucleotide polymorphisms (SNPs), RNA secondary structure, protein-protein interaction (PPI), pathway network, differentially expressed genes (DEGs), and patient survival rate. We aimed to evaluate impact of the SNPs on progression of stomach adenocarcinoma. Results We identified 22 critical DEGs involved in cell-cell and cell-matrix interaction. Seven of these key DEGs, including COL12A1, were associated with poorer overall survival. Interestingly, we found that COL12A1 was targeted by the miRNAs: hsa-miR-130b-5p (which carries the mutations rs1174814901 and rs761813865) and hsa-miR-18a-5p (which carries the mutation rs1198460870). These mutations lead to a change in the secondary structure of the miRNAs. And we also found that the miRNA target sites of COL12A1 were also mutated. These mutations at both miRNAs and COL12A1 likely attenuated their binding capability, leading to compensatory miRNA overexpression in the STAD patients. The miRNA overexpression was further found to be significantly associated with worse survival in STAD patients. Conclusions Our results suggest that mutations in Col12A1 and its miRNAs promote STAD via loss of COL12A1 suppression, providing new insight into the molecular mechanisms of STAD progression. This finding has significant implications for the development of novel noninvasive diagnostic, prognostic and therapeutic biomarkers.

Suppl. Table 1. Origin of cancer cell lines used in this study. Suppl. Table 2. Quantitative HTS screen in PANC1 cells grown as spheroids. Results of full ten concentration drug-response curves of 1,912 individual agents and compounds in PANC1 spheroids. SID, Sample ID; CCLASS, Curve class; CCLASS2, Curve class (latest version); LAC50, log AC50 (M); HILL, Hill coefficient; ZERO, fitted response at minimum concentration; INF, Fitted response at maximum concentration; MAXR, Observed response at maximum concentration; DATA0 - DATA10, Responses; C0 - C10, Doses (in µM); auc, Area Under the Curve (via integration of the fit). This data is available via the PubChem database (AID 1259363). Suppl. Table 3 Top twenty hits of KiNativ profiling of PANC1 cells with at least {greater than or equal to}50 percent inhibition of ADP binding following treatment with 500 nM of inhibitor #1. Suppl. Table 4. In vitro activity of inhibitor #1 against a panel of over 400 kinases (DiscoveRx's KinomeScan) and in situ activity of inhibitor #1 in PANC1 spheroid cells (KiNative). Suppl. Table 5. Outcomes of both 6Ã-6 and 10Ã-10 combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs.

Suppl. Fig. 1: Procedures for synthesis of NCGC00188382 (Inhibitor#1) Suppl. Fig. 2: qPCR array of 96 stem cell genes from PANC1 adherent and PANC1 spheroids reveal up-regulation of six stem cell genes in organotypic Panc1 spheres. Suppl. Fig. 3: Inhibitor #1 induces apoptosis in KLM-1 spheroids. Flow cytometry measuring fraction of cells with exposed extracellular phosphatidylserine via annexin V-FITC (in PI stained cells; y-axis). Suppl. Fig. 4: In vivo activity of inhibitor #1. Suppl. Fig. 5: Inhibitor #1 suppresses downstream signaling of aurora kinase B, and ephrin B2 receptor activation identified as targets within in situ kinome screen. Suppl. Fig. 6: Outcomes of combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs. Suppl. Fig. 7: Confirmation of synergies derived from 10Ã-10 matrix screening with inhibitor #1 in PANC1 spheroids. Suppl. Fig. 8: A. Flow cytometry measuring DNA breaks and fragments after labeling of 3'-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-UTP) after 96-hour treatment with #1 in PANC1 spheroids and 2D monolayer cells.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell–like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine–based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo. An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Suppl. Table 1. Origin of cancer cell lines used in this study. Suppl. Table 2. Quantitative HTS screen in PANC1 cells grown as spheroids. Results of full ten concentration drug-response curves of 1,912 individual agents and compounds in PANC1 spheroids. SID, Sample ID; CCLASS, Curve class; CCLASS2, Curve class (latest version); LAC50, log AC50 (M); HILL, Hill coefficient; ZERO, fitted response at minimum concentration; INF, Fitted response at maximum concentration; MAXR, Observed response at maximum concentration; DATA0 - DATA10, Responses; C0 - C10, Doses (in µM); auc, Area Under the Curve (via integration of the fit). This data is available via the PubChem database (AID 1259363). Suppl. Table 3 Top twenty hits of KiNativ profiling of PANC1 cells with at least {greater than or equal to}50 percent inhibition of ADP binding following treatment with 500 nM of inhibitor #1. Suppl. Table 4. In vitro activity of inhibitor #1 against a panel of over 400 kinases (DiscoveRx's KinomeScan) and in situ activity of inhibitor #1 in PANC1 spheroid cells (KiNative). Suppl. Table 5. Outcomes of both 6Ã-6 and 10Ã-10 combination drug screening of inhibitor #1 versus a collection of approved and investigational drugs.

Herpes simplex viruses (HSVs) often cause latent infection for a lifetime, leading to repeated recurrence. HSVs have been engineered as oncolytic HSVs. The mechanism of the latent infection and recurrence remains largely unknown, which brings great challenges and limitations to eliminate HSVs in clinic and engineer safe oHSVs. Here, we systematically reviewed the latest development of the multi-step complex process of HSV latency and reactivation. Significantly, we first summarized the three HSV latent infection pathways, analyzed the structure and expression of the LAT1 and LAT2 of HSV-1 and HSV-2, proposed the regulation of LAT expression by four pathways, and dissected the function of LAT mediated by five LAT products of miRNAs, sRNAs, lncRNAs, sncRNAs and ORFs. We further analyzed that application of HSV LAT deletion mutants in HSV vaccines and oHSVs. Our review showed that deleting LAT significantly reduced the latency and reactivation of HSV, providing new ideas for the future development of safe and effective HSV therapeutics, vaccines and oHSVs. In addition, we proposed that RNA silencing or RNA interference may play an important role in HSV latency and reactivation, which is worth validating in future.

Tumor-initiating cells (TICs) or cancer stem cells are believed to be responsible for gastrointestinal tumor initiation, progression, metastasis, and drug resistance. It is hypothesized that gastrointestinal TICs (giTICs) might originate from cell-cell fusion. Here, we systemically evaluate the evidence that supports or opposes the hypothesis of giTIC generation from cell-cell fusion both in vitro and in vivo. We review giTICs that are capable of initiating tumors in vivo with 5000 or fewer in vivo fused cells. Under this restriction, there is currently little evidence demonstrating that giTICs originate from cell-cell fusion in vivo. However, there are many reports showing that tumor generation in vitro occurs with more than 5000 fused cells. In addition, the mechanisms of giTIC generation via cell-cell fusion are poorly understood, and thus, we propose its potential mechanisms of action. We suggest that future research should focus on giTIC origination from cell-cell fusion in vivo, isolation or enrichment of giTICs that have tumor-initiating capabilities with 5000 or less in vivo fused cells, and further clarification of the underlying mechanisms. Our review of the current advances in our understanding of giTIC origination from cell-cell fusion may have significant implications for the understanding of carcinogenesis and future cancer therapeutic strategies targeting giTICs.

Clinical studies have shown that melatonin lowers the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherapy. Here, we investigated the mechanisms by which melatonin promotes platelet formation and survival. Our results show that melatonin exerted protective effects on serum-free induced apoptosis of CHRF megakaryocytes (MKs). Melatonin promoted the formation of MK colony forming units (CFUs) in a dose-dependent manner. Using doxorubicin-treated CHRF cells, we found that melatonin rescued G2/M cell cycle arrest and cell apoptosis induced by doxorubicin. The expression of p-AKT was increased by melatonin treatment, an effect that was abolished by melatonin receptor blocker. In addition, we demonstrated that melatonin enhanced the recovery of platelets in an irradiated mouse model. Megakaryopoiesis was largely preserved in melatonin-treated mice. We obtained the same results in vivo from bone marrow histology and CFU-MK formation assays. Melatonin may exert these protective effects by directly stimulating megakaryopoiesis and inhibiting megakaryocyte apoptosis through activation of its receptors and AKT signaling.

Umbilical cord blood transplantation was first reported in 1980. Since then, additional research has indicated that umbilical cord blood stem cells (UCBSCs) have various advantages, such as multi-lineage differentiation potential and potent renewal activity, which may be induced to promote their differentiation into a variety of seed cells for tissue engineering and the treatment of clinical and metabolic diseases. Recent studies suggested that UCBSCs are able to differentiate into nerve cells, chondrocytes, hepatocyte-like cells, fat cells and osteoblasts. The culture of UCBSCs has developed from feeder-layer to feeder-free culture systems. The classical techniques of cell labeling and tracing by gene transfection and fluorescent dye and nucleic acid analogs have evolved to DNA barcode technology mediated by transposon/retrovirus, cyclization recombination-recombinase and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 strategies. DNA barcoding for cell development tracing has advanced to include single cells and single nucleic acid mutations. In the present study, the latest research findings on the development and differentiation, culture techniques and labeling and tracing of UCBSCs are reviewed. The present study may increase the current understanding of UCBSC biology and its clinical applications.

Herpes simplex virus (HSV) infection in the human body can be latent in neurons for long time and be reactivated leading to recurrence at high rate. Currently there is no effective clinical strategy for the prevention and treatment of the disease relapse. HSV LAT gene is expressed in large quantities and lytic genes are turned off leading to HSV latency. Disruption of the gene expression is thought to cause HSV reactivation and disease relapse. To reveal the essence of HSV latency and reactivation, we summarized and innovatively classified the role, mechanism and transcriptional regulation of LAT in HSV latency and reactivation. This review may have important implications for future studies on HSV latency and reactivation, HSV disease prevention and treatment, and safer and more effective oncolytic HSVs (oHSVs).

Long non-coding RNAs (lncRNAs) are a novel class of gene regulators playing multifaceted roles in physiological processes as well as pathological conditions such as cancer. Cancer stem cells (CSCs) are a small subset of tumor cells that constitute the origin and development of various malignant tumors. CSCs have been identified in a wide spectrum of human tumors and could act as a critical link underlying the processes of tumor metastasis and recurrence. Mounting evidence indicates that lncRNAs are aberrantly expressed in diverse CSCs and regulate CSC properties at different molecular levels. Here, we very briefly summarize the recent findings on the potential roles of lncRNAs in regulating various functions of CSCs, and elaborate on how can lncRNAs impact CSC properties via interacting with other macromolecules at the epigenetic, transcriptional, and post-transcriptional levels. This mini-review also highlights the understanding of the modular regulatory principles of lncRNA interactions in CSCs.

Background: Drynaria Fortunei and Cuscuta Chinensis are among the most used traditional Chinese medicine herbal prescriptions and have a significant therapeutic effect on osteoarthritis. However, the purpose of this study intends to elaborate the molecular mechanism of action through network pharmacology. The active ingredients of TCM and the potential targets for the treatment of osteoarthritis were selected through the TCMSP, OMIM and Genecards. Results: The 27 components and 85/117 targets of Drynaria Fortunei and/or Cuscuta Chinensis were identified for osteoarthritis. Pharmacological and PPI network analysis identified top 3 active components (kaempferol, luteolin, and quercetin) and core proteins (IL6, AKT1, and VEGFA). GO and KEGG analysis identified the top 3 functions (cytokine and cell/nuclear receptor) and pathways (PI3K-Akt, TNF and IL-17). Molecular docking showed strong binding ability between quercetin-AKT1 and luteolin-IL6/VEGFA. Interaction analysis mapped the quercetin-AKT1 and luteolin-IL6/VEGFA binding to specific hydrogen and hydrophobic bonds. Conclusions: The main active components, common target proteins, functional activities, and signaling pathways of TCM Drynaria Fortunei and Cuscuta Chinensis for the treatment of osteoarthritis were identified by Network pharmacology. We found, for the first time, that drynariae rhizoma and cuscuta chinensis suppress osteoarthritis by quercetin-AKT1/IL6 and luteolin-VEGFA direct binding. Our findings have significant implication for our understanding of the molecular mechanism of action in the treatment of osteoarthritis and future development of osteoarthritis treatment using quercetin and luteolin.

Breast cancer is the most diagnosed cancer in women. It significantly impairs a patient's physical and mental health. Gut microbiota comprise the bacteria residing in a host's gastrointestinal tract. Through studies over the last decade, we now know that alterations in the composition of the gut microbiome are associated with protection against colonization by pathogens and other diseases, such as diabetes and cancer. This review focuses on how gut microbiota can affect breast cancer development through estrogen activity and discusses the types of bacteria that may be involved in the onset and the progression of breast cancer. We also describe potential therapies to curtail the risk of breast cancer by restoring gut microbiota homeostasis and reducing systemic estrogen levels. This review will further explore the relationship between intestinal microbes and breast cancer and propose a method to treat breast cancer by improving intestinal microbes. We aimed at discovering new methods to prevent or treat BC by changing intestinal microorganisms.

An oncolytic herpes simplex virus (oHSV) has proven amenable in oncolytic virotherapy and was approved to treat melanoma. The immediate-early (IE) protein ICP27 encoded by gene UL54 is essential for HSV infection. Post-transcriptional modification of UL54 would increase tumor targeting of oHSVs. However, UL54 gene transcription regulatory sequences and factors were not reported yet. Here we isolated a new strain LXMW of type 1 HSV (HSV-1-LXMW) in China and found it's closely related to HSV-1 strains Patton and H129 in the US by the first and next generation DNA sequencing viral DNA phylogenetic analysis. Using a weight matrix-based program Match, we found the UL54 transcription regulatory sequences binding to the transcription factors Oct-1, v-Myb and Pax-6 in HSV-1-LXMW, while the sequences binding to Oct-1 and Hairy in a HSV-2 strain. Further validation showed that HSV-1 and HSV-2 shared the common sequence binding to Oct-1, but had unique sequences to bind v-Myb and Pax-6, or Hairy, respectively, by DNA sequence alignment of total 11 HSV strains. The published results howed that the expression of transcription factors is consistent with the tissue tropism of HSV-1 and HSV-2. In the current article a new HSV-1 strain LXMW was isolated and its putative HSV UL54 transcription regulatory sequences and factors were identified for the first time. Our findings highlight the new understanding of the principles of transcriptional regulation in HSV biology and oncolytic virotherapy.

Background Herpes simplex virus (HSV) can cause encephalitis. Its infected cell polypeptide 47 (ICP47), encoded by immediate-early gene US12, promotes immune escape. ICP47 was modified in the clinically approved oncolytic HSV (oHSV) T-Vec. However, transcription regulatory sequence (TRS) and transcription regulatory factor (TRF) of HSV US12 are seldom reported. Methods Previously, our laboratory isolated a new HSV strain named HSV-1-LXMW from a male patient with oral herpes in Beijing, China. Firstly, the genetic tree was used to analyze its genetic relationship. The US12 TRS and TRF in HSV-1-LXMW were found by using predictive software. Secondly, the further verification by the multi-sequence comparative analysis shown that the upstream DNA sequence of HSV US12 gene contained the conserved region. Finally, the results of literature search shown that the expression of transcription factors was related to the tissue affinity of HSV-1 and HSV-2, so as to increase the new understanding of the transcriptional regulation of HSV biology and oncolytic virus (OVs) therapy. Results Here we reported the transcriptional regulation region sequence of our new HSV-1-LXMW, and its close relationship with HSV-1-CR38 and HSV-1-17. Importantly we identified eight different kinds of novel TRSs and TRFs of HSV US12 for the first time, and found they are conserved among HSV-1 (c-Rel, Elk-1, Pax-4), HSV-2 (Oct-1, CF2-II, E74A, StuAp) or both HSVs (HNF-4). The TRFs c-Rel and Oct-1 are biologically functional respectively in immune escape and viral replication during HSV infection. Conclusions Our findings have important implication to HSV biology, infection, immunity and oHSVs.

Herpes simplex viruses (HSVs) are important pathogens and ideal for gene therapy due to its large genome size. Previous researches on HSVs were hampered because the technology to construct recombinant HSVs were based on DNA homology-dependent repair (HDR) and plaque assay, which are inefficient, laborious, and time-consuming. Fortunately, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) recently provided the possibility to precisely, efficiently, and rapidly edit genomes and indeed is successfully being used in HSVs. Importantly, CRISPR/Cas9 technology increased HSV HDR efficiency exponentially by a 10,000-1,000,000 times when making recombinant HSVs, and its combination with flow cytometric technology made HSV recombination practically automatic. These may have a significant impact on virus and gene therapy researches. This review will summarize the latest development and molecular mechanisms of CRISPR/Cas9 genome editing technology and its recent application in HSVs.

Oncolytic herpes simplex viruses (oHSVs) have been approved for clinical usage and become more and more popular for tumor virotherapy. However, there are still many issues for the oHSVs used in clinics and clinical trials. The main issues are the limited anti-tumor effects, intratumor injection, and some side effects. To overcome such challenges, here we review the genetic engineering of the envelope glycoproteins for oHSVs to target tumors specifically, and at the same time we summarize the many neutralization antibodies against the envelope glycoproteins and align the neutralization epitopes with functional domains of the respective glycoproteins for future identification of new functions of the glycoproteins and future engineering of the epitopes to escape from host neutralization.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) contain 12 family members(CEACAM1、CEACAM3、CEACAM4、CEACAM5、CEACAM6、CEACAM7、CEACAM8、CEACAM16、CEACAM18、CEACAM19、CEACAM20 and CEACAM21)and are expressed diversely in different normal and tumor tissues. CEA (CEACAM5) has been used as a tumor biomarker since 1965. Here we review the latest research and development of the structures, expression, and function of CEACAMs in normal and tumor tissues, and their application in the tumor diagnosis, prognosis, and treatment. We focus on recent clinical studies of CEA targeted cancer immunotherapies, including bispecific antibody (BsAb) for radio-immuno-therapy and imaging, bispecific T cell engager (BiTE) and chimeric antigen receptor T cells (CAR-T). We summarize the promising clinical relevance and challenges of these approaches and give perspective view for future research. This review has important implications in understanding the diversified biology of CEACAMs in normal and tumor tissues, and their new role in tumor immunotherapy.

Herpes simplex viruses (HSVs) cause cold sores and genital herpes and can establish lifelong latent infection in neurons. An engineered oncolytic HSV (oHSV) has recently been approved to treat tumors in clinics. HSV latency-associated transcripts (LATs) are associated with the latent infection, but LAT transcriptional regulation was seldom reported. For a better treatment of HSV infection and tumors, here we sequenced the LAT encoding DNA and LAT transcription regulatory region of our recently isolated new strain HSV-1-LXMW and did comparative analysis of the sequences together with those of other four HSV-1 and two HSV-2 strains. Phylogenetic analysis of LATs revealed that HSV-1-LXMW is evolutionarily close to HSV-1-17 from MRC University, Glasgow, UK. For the first time, Using a weight matrix-based program Match and multi-sequences alignment of the 6 HSV strains, we identified HSV LAT transcription regulatory sequences that bind to 9 transcription factors: AP-1, C-REL, Comp1, E2F, Hairy, HFH-3, Kr, TCF11/MAFG, v-Myb. Interestingly, these transcription regulatory sequences and factors are either conserved or unique among LATs of HSV-1 and HSV-2, suggesting they are potentially functional. Furthermore, literature analysis found that the transcription factors v-myb and AP-1 family member JunD are functional in regulating HSV gene transcription, including LAT transcription. For the first time, we discovered seven novel transcription factors and their corresponding transcription regulatory sequences of HSV LATs. Based on our findings and other reports, we proposed potential mechanisms of the initiation and maintenance of HSV latent infection. Our findings may have significant implication in our understanding of HSV latency and engineering of better oncolytic HSVs.

Long non-coding RNAs (lncRNAs) are versatile regulators of gene expression and play crucial roles in diverse biological processes. Epithelial-mesenchymal transition (EMT) is a cellular program that drives plasticity during embryogenesis, wound healing, and malignant progression. Increasing evidence shows that lncRNAs orchestrate multiple cellular processes by modulating EMT in diverse cell types. Dysregulated lncRNAs that can impact epithelial plasticity by affecting different EMT markers and target genes have been identified. However, our understanding of the landscape of lncRNAs important in EMT is far from complete. Here, we summarize recent findings on the mechanisms and roles of lncRNAs in EMT and elaborate on how lncRNAs can modulate EMT by interacting with RNA, DNA, or proteins in epigenetic, transcriptional, and post-transcriptional regulation. This review also highlights significant EMT pathways that may be altered by diverse lncRNAs, thereby suggesting their therapeutic potential.

Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiation‑induced myelosuppressive mouse model. Colony‑forming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and anti‑apoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day 21 following treatment, as well as platelets and white blood cells at day 14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that tri‑lineage hematopoiesis was preserved in the ASPS‑ and thrombopoietin (TPO)‑treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPS‑treated group was similar to that of the TPO‑treated group. Additionally,emin vitro/emexperiments indicated that treatment with 100 emµ/emg/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase‑3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell–like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine–based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo. An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Background & Aims: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including liver. It's important to know if LRCC, a novel class of CSC, are relatively resistant to metformin, unlike other types of CSC. As metformin inhibits the Sorafenib-Target-Protein (STP) PI3K, and LRCC are newly described CSC, we undertook this study to test the effects of Metformin on Sorafenib-treated HCC and HCC-derived-LRCC. Methods: We tested various STP levels and phosphorylation status, associated genes' expression, proliferation, viability, toxicity, and apoptosis profiles, before and after treatment with Sorafenib with/without Metformin. Results: Metformin enhances the effects of Sorafenib on HCC, and significantly decreased viability/proliferation of HCC cells. This insulin-independent effect was associated with inhibition of multiple STPs (PKC, ERK, JNK and AKT). However, Metformin increased the relative proportion of LRCCs. Comparing LRCC vs. non-LRCC, this effect was associated with improved toxicity and apoptosis profiles, down-regulation of cell death genes and up-regulation of cell proliferation and survival genes in LRCC. Concomitantly, Metformin up-regulated pluripotency, Wnt, Notch and SHH pathways genes in LRCC vs. non-LRCC. Conclusions: Metformin and Sorafenib have enhanced anti-cancer effects. However, in contradistinction to reports on other types of CSC, Metformin is less effective against HCC-derived-CSC LRCC. Our results suggest that combining Metformin with Sorafenib may be able to repress the bulk of tumor cells, but as with other anti-cancer drugs, may leave LRCC behind leading to cancer recurrence. Therefore, liver LRCC, unlike other types of CSC, are relatively resistant to the reported anti-cancer stem cell drug metformin. This is the first report that there is a type of CSC that is not relatively resistant to the CSC-targeting drug. Our findings suggest that a drug targeting LRCC may be critically needed to target CSC and prevent cancer recurrence. These may significantly contribute to the understanding of Metformin's anti-cancer effects and the development of novel drugs targeting the relatively resistant LRCC.

In 2003 in China, Peng et al. invented the recombinant adenovirus expressing p53 (Gendicine) for clinical tumor virotherapy. This was the first clinically approved gene therapy and tumor virotherapy drug in the world. An oncolytic herpes simplex virus expressing granulocyte-macrophage colony-stimulating factor (Talimogene laherparepvec) was approved for melanoma treatment in the United States in 2015. Since then, oncolytic viruses have been attracting more and more attention in the field of oncology, and may become novel significant modalities of tumor precision imaging and radiotherapy after further improvement. Oncolytic viruses carrying reporter genes can replicate and express genes of interest selectively in tumor cells, thus improving in vivo noninvasive precision molecular imaging and radiotherapy. Here, the latest developments and molecular mechanisms of tumor imaging and radiotherapy using oncolytic viruses are reviewed, and perspectives are given for further research. Various types of tumors are discussed, and special attention is paid to gastrointestinal tumors.

Recently, we reported that anticancer bioactive peptide (ACBP), purified from goat spleens immunized with human gastric cancer extracts, significantly inhibited gastric cancer cells in vitro and gastric tumors in vivo via repressing cell growth and promoting apoptosis, making it a promising potential biological anticancer drug. However, it is not known what genes are functionally required for the ACBP effects. Here, we first found that two tumor suppressor genes, cyclin-dependent kinase inhibitor 2B (CDKN2B) and growth arrest and DNA damage-inducible alpha (GADD45A), were upregulated significantly in the cells with ACBP treatment by microarray screening and the findings were validated by real-time RT-PCR. Next, GADD45A mRNA and protein expressions were downregulated in the gastric cancer cells by lentivirus-mediated RNAi; then, cell viability, cell cycle, and apoptosis were assayed by MTT and flow cytometry. Interestingly, our results indicated that cell viability was not dependent on GADD45A without ACBP treatment; however, cell sensitivity to ACBP was significantly decreased in ACBP-treated gastric cancer cells with GADD45A downregulation. Therefore, we demonstrate that GADD45A was functionally required for ACBP to inhibit gastric cancer cells, suggesting that GADD45A may become a biomarker for ACBP sensitivity. Our findings have significant implications on the molecular mechanism understanding, biomarker development, and anticancer drug development of ACBP.

Objective The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib. Methods We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib. Results LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. Conclusions Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.

Objective: Stem-like cancer cells contribute to cancer initiation and maintenance. Stem cells can self-renew by asymmetric cell division (ACD). ACD with non-random chromosomal cosegregation (ACD-NRCC) is one possible self-renewal mechanism. There is a paucity of evidence supporting ACD-NRCC in human cancer. Our aim was to investigate ACD-NRCC and its potential interactions with the cancer niche (microenvironment) in gastrointestinal cancers. Design: We used DNA double and single labeling approaches with FACS to isolate live cells undergoing ACD-NRCC. Results: Gastrointestinal cancers contain rare subpopulations of cells capable of ACD-NRCC. ACD-NRCC was detected preferentially in subpopulations of cells previously suggested to be stem-like/tumor-initiating cancer cells. ACD-NRCC was independent of cell-to-cell contact, and was regulated by the cancer niche in a heat-sensitive paracrine fashion. Wnt pathway genes and proteins are differentially expressed in cells undergoing ACD-NRCC vs. symmetric cell division. Blocking the Wnt pathway with IWP2 (WNT antagonist) or siRNA-TCF4 resulted in suppression of ACD-NRCC. However, using a Wnt-agonist did not increase the relative proportion of cells undergoing ACD-NRCC. Conclusion: Gastrointestinal cancers contain subpopulations of cells capable of ACD-NRCC. Here we show for the first time that ACD-NRCC can be regulated by the Wnt pathway, and by the cancer niche in a paracrine fashion. However, whether ACD-NRCC is exclusively associated with stem-like cancer cells remains to be determined. Further study of these findings might generate novel insights into stem cell and cancer biology. Targeting the mechanism of ACD-NRCC might engender novel approaches for cancer therapy.

No systemic therapy is effective against pancreatic cancer (PC). Pancreatic cancer stem cells (PCSC) are hypothesized to account for therapeutic resistance. Several PCSC subpopulations were reported, each characterized by different markers. To be able to target PCSC, we sought to better define this putative heterogeneity. Therefore, we tested most of the known putative PCSC markers in established and fresh tumor cell lines. CD20, CD24, CD44, CD133, CD184 (CXCR4), CD326 (EpCam, ESA), Sox-2, OCT 3/4, and the side-population (SP) were tested in five PC cell lines, and the effects of confluency, hypoxia, radiation, and gemcitabine on the SP. The testing phase suggested several putative PCSC populations that were further tested and validated for their tumor-initiating capacity against known PCSC in 3 established and 1 fresh PC cell lines. Cell surface and intracellular markers showed significant variability among cell lines. SP was the only common marker in all cell lines and consistently less than 1%. SP response to confluence, hypoxia, radiation, and gemcitabine was inconsistent between cell lines. The initial testing phase suggested that SP/CD44-CD24-CD326+ cells might be a novel PCSC subpopulation. Tumor initiation capacity tests in nude mice confirmed their increased tumorigenicity over previously reported PCSC. Our data better define the heterogeneity of reported PCSC in cell lines tested in this study. We propose that prior to targeting PC via PCSC, one will need to gain more insight into this heterogeneity. Finally, we show that SP/CD44-CD24-CD326+ cells are a novel subpopulation of pancreatic cancer tumor initiating cells. Further mechanistic studies may lead to better targeting of PC via targeting this novel PCSC.

Colorectal cancer (CRC) holds the third highest incidence and cancer related mortality rate among men and women in the United States. Unfortunately, there has been little progression made in the treatment of this deadly disease once it has spread beyond the colon. It has been hypothesize that colon cancer stem cells are implicated in CRC carcinogenesis, metastasis, and therapeutic resistance. One of the difficulties in testing these hypotheses is the current use of established high-passage cancer cell lines. Long term, high-passage established cell lines have cells with stem like properties as they propagate almost indefinitely. These cells are thought to be different than the original cancer stem cells in fresh tumors. In order to investigate cancer stem cells, and molecularly profiling tumors with high fidelity to the original primary tumor, one needs to establish suitable primary ultra-low passage cell lines from fresh surgical specimens. Here we report the establishment of tumor initiating colon cancer ultra-low passage cell lines by a combination of gentle mechanical, enzymatic dissociation, spheroid formation, and followed by two generation xenografts from fresh tumors obtained at time of operation. Tumors generated were characterized by morphology, flow cytometry, immunofluorescence, and by gene expression. In the future, such a technology can be used to produce expeditiously enough material to test for mutations, genetic signatures and molecular subtyping readily available for clinical therapeutic decision making.

Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. Disclosure of potential conflicts of interest is found at the end of this article.

Introduction: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression.Methods: Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = Results: Of 30 patients 7 were AJCC stage IIA, 10 stage IIIB, 7 stage IIIC and 6 stage IV. Median follow-up was 113 months. DFI was 17 months. Median overall survival (OS) was not reached. Stage-specific OS was: II - not reached; III - not reached; IV - 11 months. In a univariate analysis, poor OS was associated with loss of CD29 expression; median OS, 32 months vs. not reached for CSS 3-7 vs. >7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014).Conclusion: CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.

Colorectal cancer (CRC) holds the third highest incidence and cancer related mortality rate among men and women in the United States. Unfortunately, there has been little progression made in the treatment of this deadly disease once it has spread beyond the colon. It has been hypothesize that colon cancer stem cells are implicated in CRC carcinogenesis, metastasis, and therapeutic resistance. One of the difficulties in testing these hypotheses is the current use of established high-passage cancer cell lines. Long term, high-passage established cell lines have cells with stem like properties as they propagate almost indefinitely. These cells are thought to be different than the original cancer stem cells in fresh tumors. In order to investigate cancer stem cells, and molecularly profiling tumors with high fidelity to the original primary tumor, one needs to establish suitable primary ultra-low passage cell lines from fresh surgical specimens. Here we report the establishment of tumor initiating colon cancer ultra-low passage cell lines by a combination of gentle mechanical, enzymatic dissociation, spheroid formation, and followed by two generation xenografts from fresh tumors obtained at time of operation. Tumors generated were characterized by morphology, flow cytometry, immunofluorescence, and by gene expression. In the future, such a technology can be used to produce expeditiously enough material to test for mutations, genetic signatures and molecular subtyping readily available for clinical therapeutic decision making.

The ability to retain DNA labels over time is a property proposed to be associated with adult stem cells. Recently, label retaining cells (LRC) were indentified in cancer. LRC were suggested to be the result of either slow-cycling or asymmetric-cell-division with nonrandom-chromosomal-cosegregation (ACD-NRCC). ACD-NRCC is proposed to segregate the older template DNA strands into daughter stem cells and newly synthesized DNA into daughter cells destined for differentiation. The existence of cells undergoing ACD-NRCC and the stem-like nature of LRC remain controversial. Currently, to detect LRC and ACD-NRCC, cells need to undergo fixation. Therefore, testing the stem-cell nature and other functional traits of LRC and cells undergoing ACD-NRCC has been limited. Here, we show a method for labeling DNA with single and dual-color nucleotides in live human liver cancer cells avoiding the need for fixation. We describe a novel methodology for both the isolation of live LRC and cells undergoing ACD-NRCC via fluorescence-activated cell sorting with confocal microscopy validation. This has the potential to be a powerful adjunct to stem-cell and cancer research.

Detection of circulating tumor cells remains a significant challenge due to their vast physical and biological heterogeneity. We developed a cell-surface-marker-independent technology based on telomerase-specific, replication-selective oncolytic herpes-simplex-virus-1 that targets telomerase-reverse-transcriptase-positive cancer cells and expresses green-fluorescent-protein that identifies viable CTCs from a broad spectrum of malignancies. Our method recovered 75.5-87.2% of tumor cells spiked into healthy donor blood, as validated by different methods, including single cell sequencing. CTCs were detected in 59-100% of 326 blood samples from patients with 6 different solid organ carcinomas and lymphomas. Significantly, CTC-positive rates increased remarkably with tumor progression from N0M0, N+M0 to M1 in each of 5 tested cancers (lung, colon, liver, gastric and pancreatic cancer, and glioma). Among 21 non-small cell lung cancer cases in which CTC values were consecutively monitored, 81% showed treatment-related decreases, which was also found after treatments in the other solid tumors. Moreover, monitoring CTC values provided an efficient treatment response indicator in hematological malignancies. Compared to CellSearch, our method detected significantly higher positive rates in 40 NSCLC in all stages, including N0M0, N+M0 and M1, and was less affected by chemotherapy. This simple, robust and clinically-applicable technology detects viable CTCs from solid and hematopoietic malignancies in early to late stages, and significantly improves clinical detection and treatment prognostication.

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has limited anticancer efficacy in EGFR-positive esophageal cancer (EsC) and malignant mesothelioma (MPM). The underlying molecular mechanism of resistance to EGFR-TKI in these types of cancer remains unclear.We tested sensitivity to EGFR-TKI, expression/activity of common signal transduction pathways and epithelial to mesenchymal transition (EMT) gene signatures in 14 EsC and MPM cultured cell lines in vitro.More than 50% EGFR-positive EsC and MPM cells were resistant to EGFR-TKI, and susceptibility to EGFR-TKI growth-inhibitory effect correlated positively with expression of E-cadherin (epithelial gene marker) and negatively with mesenchymal gene markers. Acquired resistance to EGFR-TKI in intrinsically sensitive cancer cells coincided with spontaneous loss of E-cadherin, while ectopic expression of E-cadherin sensitized resistant cells to EGFR-TKI.E-Cadherin expression appears to be not only a strong biomarker but also a functional requirement and potential therapeutic target for sensitivity to EGFR-TKI.

Background: Pancreatic cancer is highly resistant to existing chemotherapy regimens, with only a minority of patients exhibiting a response to standard therapy. One aspect of treatment resistance is based on the cancer stem cell (CSC) hypothesis, which holds that a small population of highly chemoresistant cells is responsible for initiating and sustaining tumor growth. Compounds with activity against this population are highly desired in the adjuvant setting, where failure to sterilize micrometastases not detected at time of operation results in recurrence in nearly all patients. Methods: A high-throughput drug screen (HTS) was performed in an in vitro model of pancreatic cancer stem-like cells (spheroids). Molecules with high potency against both the cancer stem cell-like spheroid and monolayer cell formats were evaluated for efficacy and mechanism of action in a variety of preclinical models of pancreatic cancer. Unbiased global in situ phosphoproteomic profiling using KiNativ technology was used to identify kinase targets impacted by the top hit. Stemness phenotype was interrogated by qRT-PCR of stemness genes, flow cytometry, colony formation assays, and tumor initiation in immunocompromised mice. Results: The top hit from the HTS was a 1H-benzo[d]imidazol-2-amine-based inhibitor of interleukin-2-inducible T-cell kinase (ITK) (NCGC00188382, 1). 1 effectively mediated growth inhibition, blocked migration and invasion, and reduced clonogenicity of cancer cells with an associated decrease in the expression of several CSC-associated stemness markers at non-lethal concentrations (Sox2, Notch1) compared to gemcitabine used at >10-fold concentration. Metastatic burden in both the liver and lung was significantly reduced in mice orthotopically injected with pancreatic CSCs in the treatment group after 6 weeks. Target deconvolution of 1 demonstrated inhibition of multiple select kinase targets. There were 16 targets identified from an initial screening panel of >400 kinases. The targets were further evaluated for the ability to phenocopy the effect of treatment with 1 via shRNA knockdown. Inhibition of the thousand-and-one amino acid kinase 3 (TAOK3), cyclin-dependent kinase 7 (CDK7), aurora B kinase (AURKB), and ephrin B2 receptor (EPHB2) effectively phenocopied the effect of treatment with 1. Concurrent silencing of two or more targets increased apoptosis induced by 1, suggesting intrinsic additivity of the multikinase agent. Enforced overexpression of TAOK3 increased expression of stemness markers, clonogenicity, and tumor initiation in vivo. Conclusion: A potent inhibitor of ITK with a unique target profile and anti-stemness activities was derived from a differential HTS in a pancreatic spheroid CSC model. The 1H-benzo[d]imidazol-2-amine-derivative targets select characteristics attributed to pancreatic CSCs, including cell migration, colony formation, and metastasis formation with concomitant suppression of stemness traits without inducing cell death at the concentrations used. Target deconvolution of the compound identified multiple kinase targets, including TAOK3, a kinase not previously known to be involved in pancreatic tumor biology. Overexpression of TAOK3 results in an enforced CSC phenotype including increased colony formation and tumor initiation in vivo. The observed inhibitory effect of 1 on stemness phenotype at nanomolar concentrations without affecting cell viability or showing toxicity in vivo supports further preclinical development. Citation Format: Taylor Aiken, Yansong Bian, Yaroslav Teper, Lesley Mathews Griner, Rajarshi Guha, Paul Shinn, Hong-Wu Xin, Holger Pflicke, Jian-kang Jiang, Paresma Patel, Steven Rogers, Jeffery Aube, Marc Ferrer, Craig J. Thomas, Udo Rudloff.{Authors}. Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B36.

Introduction: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression. Methods: Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = 7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014). Conclusion: CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.